The Tumor Necrosis Factor/Nerve Growth Factor (TNF/NGF) receptor superfamily is defined by structural homology between the extracellular domains of its members (Bazan, 1993; Beutler and van Huffel, 994; Smith et al., 1994). Except for two receptors, the p55 TNF receptor and Fas/APO1, the various members of this receptor family do not exhibit clear similarity of structure in their intracellular domains. Nevertheless, there is much similarity of function between the receptors, indicating that they share common signaling pathways. One example for this similarity is the ability of several receptors of the TNF/NGF family to activate the transcription factor NF-κB. This common ability was ascribed to a capability of a cytoplasmic protein that activates NF-κB, TNF Receptor Associated Factor 2 (TRAF2) to bind to the structurally-dissimilar intracellular domains of several of the receptors of the TNF/NGF family. By what mechanisms TRAF2 acts and how its responsiveness to the different receptors to which it binds is coordinated, is not known.
TRAF2 is a member of a recently described family of proteins called TRAF that includes several proteins identified as, for example, TRAF1, TRAF2 (Rothe, M., Wong, s.c., Henzel, W. J. and Goeddel, D (1994) Cell 78:681-692; PCT published application WO 95/33051), TRAF3 (Cheng, G. et al. (1995)), and TRAF6 (see Cao et al., 1996a).
All proteins belonging to the TRAF family share high degree of amino acid identity in their C-terminal domains, while their N-terminal domains may be unrelated. As shown in a schematic illustration of TRAF2 (FIG. 1), the molecule contains a ring finger motif and two TFIIIA-like zinc finger motifs at its C-terminal area. The C-terminal half of the molecule includes a region known as the “TRAF domain” containing a potential leucine zipper region extending between amino acids 264-358 (called N-TRAF), and another part towards the carboxy end of the domain between amino acids 359-501 (called C-TRAF) which is responsible for TRAF binding to the receptors and to other TRAF molecules to form homo- or heterodimers.
Activation of the transcription factor NF-κB is one manifestation of the signaling cascade initiated by some of the TNF/NGF receptors and mediated by TRAF2. NF-κB comprises members of a family of dimer-forming proteins with homology to the Rel oncogene which, in their dimeric form, act as transcription factors. These factors are ubiquitous and participate in regulation of the expression of multiple genes. Although initially identified as a factor that is constitutively present in B cells at the stage of IgK light chain expression, NF-κB is known primarily for its action as an inducible transcriptional activator. In most known cases NF-κB behaves as a primary factor, namely the induction of its activity is by activation of pre-existing molecules present in the cell in their inactive form, rather than its de-novo synthesis which in turn relies on inducible transcription factors that turn-on the NF-κB gene. The effects of NF-κB are highly pleiotropic. Most of these numerous effects share the common features of being quickly induced in response to an extracellular stimulus. The majority of the NF-κB-activating agents are inducers of immune defense, including components of viruses and bacteria, cytokines that regulate immune response, UV light and others. Accordingly, many of the genes regulated by NF-κB contribute to immune defense (see Blank et al., 1992; Grilli et al., 1993; Baeuerle and Henkel, 1994, for reviews).
One major feature of NF-κB-regulation is that this factor can exist in a cytoplasmic non-DNA binding form which can be induced to translocate to the nucleus, bind DNA and activate transcription. This dual form of the NF-κB proteins is regulated by I-κB—a family of proteins that contain repeats of a domain that has initially been discerned in the erythrocyte protein ankyrin (Gilmore and Morin, 1993). In the unstimulated form, the NF-κB dimer occurs in association with an I-κB molecule which imposes on it cytoplasmic location and prevents its interaction with the NF-κB-binding DNA sequence and activation of transcription. The dissociation of I-κB from the NF-κB dimer constitutes the critical step of its activation by many of its inducing agents (DiDonato et al., 1995). Knowledge of the mechanisms that are involved in this regulation is still limited. There is also just little understanding of the way in which cell specificity in terms of responsiveness to the various NF-κB-inducing agents is determined.
One of the most potent inducing agents of NF-κB is the cytokine tumor necrosis factor (TNF). There are two different TNF receptors, the p55 and p75 receptors (p55-R and p75-R). Their expression levels vary independently among different cells (Vandenabeele et al., 1995). The p75 receptor responds preferentially to the cell-bound form of TNF (TNF is expressed both as a beta-transmembrane protein and as a soluble protein) while the p55 receptor responds just as effectively to soluble TNF molecules (Grell et al., 1995). The intracellular domains of the two receptors are structurally unrelated and bind different cytoplasmic proteins. Nevertheless, at least part of the effects of TNF, including the cytocidal effect of TNF and the induction of NF-κB, can be induced by both receptors. This feature is cell specific. The p55 receptor is capable of inducing a cytocidal effect or activation of NF-κB in all cells that exhibit such effects in response to TNF. The p75-R can have such effects only in some cells. Others, although expressing the p75-R at high levels, show induction of the effects only in response to stimulation of the p55-R (Vandenabeele et al., 1995). Apart from the TNF receptors, various other receptors of the TNF/NGF receptor family: CD30 (McDonald et al., 1995), CD40 (Berberich et al., 1994; Lalmanach-Girard et al., 1993), the lymphotoxin beta receptor and, in a few types of cells, Fas/APO1 (Rensing-Ehl et al., 1995), are also capable of inducing activation of NF-κB. The IL-1 type I receptor, also effectively triggering NF-κB activation, shares most of the effects of the TNF receptors despite the fact that it has no structural similarity to them.
The activation of NF-κB upon triggering of these various receptors results from induced phosphorylation of its associated I-κB molecules. This phosphorylation tags I-κB to degradation, which most likely occurs in the proteasome. The nature of the kinase that phosphorylates I-κB, and its mechanism of activation upon receptor triggering is still unknown. However, in the recent two years some knowledge has been gained as to the identity of three receptor-associated proteins that appear to take part in initiation of the phosphorylation (see diagrammatic illustration in FIGS. 2a and 6). A protein called TRAF2, initially cloned by D. Goeddel and his colleagues (Rothe et al., 1994), seems to play a central role in NF-κB-activation by the various receptors of the TNF/NGF family. The protein, which when expressed at high levels can by itself trigger NF-κB activation, binds to activated p75 TNF-R (Rothe et al., 1994), lymphotoxin beta receptor (Mosialos et al., 1995), CD40 (Rothe et al., 1995a) and CD-30 (unpublished data) and mediates the induction of NF-κB by them. TRAF2 does not bind to the p55 TNF receptor nor to Fas/APO1, however, it can bind to a p55 receptor-associated protein called TRADD and TRADD has the ability to bind to a Fas/APO1-associated protein called MORT1 (or FADD—see Boldin et al. 1995b and 1996). Another receptor-interacting protein, called RIP (see Stanger et al., 1995) is also capable of interacting with TRAF2 as well as with FAS/APO1, TRADD, the p55 TNF receptor and MORT-1. Thus, while RIP has been associated with cell cytotoxicity induction (cell death), its ability to interact with TRAF2 also implicates it in NF-κB activation and it also may serve in addition to augment the interaction between FAS/APO1, MORT-1, p55 TNF receptor and TRADD with TRAF2 in the pathway leading to NF-κB activation. These associations apparently allow the p55 TNF receptor and Fas/APO1 to trigger NF-κB activation (Hsu et al., 1995; Boldin et al., 1995; Chinnaiyan et al., 1995; Varfolomeev et al., 1996; Hsu et al., 1996). The triggering of NF-κB activation by the IL-1 receptor occurs independently of TRAF2 and may involve a recently-cloned IL-1 receptor-associated protein-kinase called IRAK (Croston et al., 1995).
By what mechanism TRAF2 acts is not clear. Several cytopiasmic molecules that bind to TRAF2 have been identified (Rothe et al., 1994; Rothe et al., 1995b). However, the information on these molecules does not provide any clue as to the way by which TRAF2, which by itself does not possess any enzymatic activity, triggers the phosphorylation of I-κB. There is also no information yet of mechanisms that dictate cell-specific pattern of activation of TRAF2 by different receptors, such as observed for the induction of NF-κB by the two TNF receptors.
In addition to the above mentioned, of the various TRAF proteins, it should also be noted that TRAF2 binds to the p55 (CD120a) and p75 (CD120b) TNF receptors, as well as to several other receptors of the TNF/NGF receptor family, either directly or indirectly via other adaptor proteins as noted above, for example with reference to the FAS/APO1 receptor, and the adaptor proteins MORT-1, TRADD and RIP. As such, TRAF2 is crucial for the activation of NF-κB (see also Wallach, 1996). However, TRAF3 actually inhibits activation of NF-κB by some receptors of the TNF/NGF family (see Rothe et al., 1995a), whilst TRAF6 is required for induction of NF-κB by IL-1 (see Cao et al., 1996a).
Accordingly, as regards NF-κB activation and its importance in maintaining cell viability, the various intracellular pathways involved in this activation have heretofore not been clearly elucidated, for example, how the various TRAF proteins, are involved directly or indirectly.
Furthermore, as is now known regarding various members of the TNF/NGF receptor family and their associated intracellular signaling pathways inclusive of various adaptor, mediator/modulator proteins (see brief reviews and references in, for example, co-pending co-owned Israel Patent Application Nos. 114615, 114986, 115319, 116588), TNF and the FAS/APO1 ligand, for example, can have both beneficial and deleterious effects on cells. TNF, for example, contributes to the defense of the organism against tumors and infectious agents and contributes to recovery from injury by inducing the killing of tumor cells and virus-infected cells, augmenting antibacterial activities of granulocytes, and thus in these cases the TNF-induced cell killing is desirable. However, excess TNF can be deleterious and as such TNF is known to play a major pathogenic role in a number of diseases such as septic shock, anorexia, rheumatic diseases, inflammation and graft-vs-host reactions. In such cases TNF-induced cell killing is not desirable. The FAS/APO1 ligand, for example, also has desirable and deleterious effects. This FAS/APO1 ligand induces via its receptor the killing of autoreactive T cells during maturation of T cells, i.e. the killing of T cells which recognize self-antigens, during their development and thereby preventing autoimmune diseases. Further, various malignant cells and HIV-infected cells carry the FAS/APO1 receptor on their surface and can thus be destroyed by activation of this receptor by its ligand or by antibodies specific thereto, and thereby activation of cell death (apoptosis) intracellular pathways mediated by this receptor. However, the FAS/APO1 receptor may mediate deleterious effects, for example, uncontrolled killing of tissue which is observed in certain diseases such as acute hepatitis that is accompanied by the destruction of liver cells.
In view of the above, namely, that receptors of the TNF/NGF family can induce cell death pathways on the one hand and can induce cell survival pathways (via NF-κB induction) on the other hand, there apparently exists a fine balance, intracellularly between these two opposing pathways. For example, when it is desired to achieve maximal destruction of cancer cells or other infected or diseased cells, it would be desired to have TNF and/or the FAS/APO1 ligand inducing only the cell death pathway without inducing NF-κB. Conversely, when it is desired to protect cells such as in, for example, inflammation, graft-vs-host reactions, acute hepatitis, it would be desirable to block the cell killing induction of TNF and/or FAS/APO1 ligand and enhance, instead, their induction of NF-κB. Likewise, in certain pathological circumstances it would be desirable to block the intracellular signaling pathways mediated by the p75 TNF receptor and the IL-1 receptor, while in others it would be desirable to enhance these intracellular pathways.
Recently, the present inventors have isolated a kinase called NIK (Israel Patent Application Nos. 117800, 119133 and WO 97/37016) which is capable of binding to TRAF2 and is directly involved in the phosphorylation reactions leading to induction of NF-κB activation.
In addition, a number of caspases have recently been isolated by a number of researchers (including the present inventors (see co-pending, co-owned Israel Patent Application No. IL 120759)), which interact with the above noted adaptor proteins (e.g. MORT-1/FADD) or with complexes between the adaptor proteins and the various receptors of the TNF/NGF receptor family and which effect the proteolytic reactions leading to apoptotic cell death. Thus, direct modulation of these caspases would be desired in the situations noted above when it is desired to inhibit or enhance cell death, for example, when it is desired to inhibit cell death it would be desirable to inhibit the activity of these caspases. In this respect it has been reported (see review in Hofmann et al., 1997) that there exists a region called a prodomain in many of these caspases that is also present in a number of adaptor proteins such as, for example, RAIDD (which interacts with RIP, TRADD and thereby with MORT-1/FADD, the p55-TNF-R and FAS/APO1), an adaptor protein of the cell death pathway; and c-IAP1, c-IAP2, two proteins which appear to be inhibitors of apoptosis and which themselves interact with TRAF2, and thereby may be inhibitors of caspases or may otherwise stimulate TRAF2 involvement in the cell survival pathway resulting in induction of NF-κB activation. As such this prodomain has also been designated as CARD for ‘caspase recruitment domain’ (see Hofmann et al., 1997). This prodomain (CARD) therefore represents another target for modulation of the intracellular signaling pathways associated with cell death induction.
Moreover, recently there has been described (see Review by Yang and Korsmeyer, 1996) another family of proteins, called the BCL2 protein family, of which the proteins BCL2, its homolog BCL-X including the two forms thereof being BCL-XL and the alternatively spliced BCL-XS, MCL1, A1, BAK, BAD, BAG1, BAX, the adenovirus E1B-19k, and the Caenorhabditis elegans (C. elegans) CED-9 protein are all members. Of these proteins it has been observed that BCL2, BCL-XL, E1B-19k and CED-9 function to inhibit apoptosis, or to protect against apoptosis induced by various intracellular signaling pathways (see Yang and Korsmeyer, 1996). BCL2 and BCL-XL are also apparently intracellular membrane-bound proteins localized to mitochondria, as well as smooth endoplasmic reticulum, and the perinuclear membrane, the C-terminus of these proteins having a signal anchor sequence responsible for targeting and insertion thereof into the outer mitochondrial membrane and the other, above noted, intracellular membranes. Once anchored in the various intracellular membranes the BCL2 and BCL-XL proteins are exposed to the cytosol where they can interact with various other intracellular proteins.
How BCL2, BCL-XL, E1B-19k and CED-9 protect cells has not yet been fully elucidated, but it appears that their effect is apparently upstream of the cell death effectors being the various caspases noted above, such as, for example ICE and ICE-like proteases of the ICE/CED-3 family including CPP32/Yama, ICE-LAP3 (Mch3), ICH-1 and others. In fact, CED-9 was found to be a specific inhibitor of the C.elegans death effector proteases CED-3 and CED-4, and BCL2 is apparently an inhibitor of ICH-1 (also called NEDD2), in particular, the ICH-IL form which promotes cell death. Thus, while the precise mechanism of inhibition of apoptosis by BCL2, BCL-XL, CED-9 and E1B-19k, is not clear, it is apparently upstream of the ICE-CED-3 proteases which are the death effectors (see review of Yang and Korsmeyer, 1996, as well as Chinnaiyan et al., 1996).
As regards the other BCL2 family members noted above, BAX is a cell death promoter. BAX binds to itself and in the form of such BAX homodimers it promotes apoptosis. BAX also binds to BCL2 and BCL-XL and such heterodimers are associated with BCL2's protective effect against apoptosis. Thus the balance between the amounts of BAX/BAX homodimers and BAX/BLC2 heterodimers determines whether cells will be susceptible to apoptosis or whether they will be protected against apoptosis. BAX is also apparently an intracellular membrane-bound protein also being localized to a large degree to the outer mitochondrial membrane (for above mentioned concerning BAX, see also review by Yang and Korsmeyer, 1996). Further, the above noted BAK and BAD proteins also act as negative regulators of BCL2 and BCL-XL activity, namely, they repress the ability of BCL2 and BCL-XL to protect cells from apoptosis. It appears that both BAK and BAD bind BCL2 and BCL-XL and thereby prevent BAX from binding to BCL2 and BCL-XL resulting in increased amounts of BAX/BAX homodimers and subsequently increased cell death (see review by Yang and Korsmeyer, 1996). In this regard it also appears that BAK functions to block the death-repressor activity of BCL2 and BCL-XL directly as BAK/BCL2 and BAK/BCL-XL heterodimers lack the ability to protect cells from apoptosis. BAD appears to act more like a competitive inhibitor for BAX binding to BCL2 and BCL-XL, as BAD may replace BAX from BAX/BCL2 and BAX/BCL-XL heterodimers, thereby providing for increased amounts of death-promoting BAX/BAX homodimers. While BAK also appears to be an intracellular membrane bound protein localized to, amongst others, mitochondrial outer membranes, BAD, however, is apparently devoid of a membrane anchor sequence and as such is not a membrane-bound protein (see review by Yang and Korsmeyer, 1996).
Another of the above members of the BCL2 family is BAG1 (see Yang and Korsmeyer, 1996) which is a positive modulator of BCL2 activity leading to enhanced BCL2 protective activity against apoptosis and even providing for BCL2 protective activity against apoptosis in cells induced to undergo apoptosis by signals not usually suppressed by BCL2.
It should also be noted that the above mentioned alternatively spliced form of BCL-XL, namely the BCL-XS protein is also an antagonist of BCL-XL and BCL2 activity, and blocks their protective activity against apoptosis (see also review of Yang and Korsmeyer, 1996).
In view of the above mentioned it appears that the BCL2 family of proteins play a role in regulating cell death or cell survival pathways intracellularly and a shift in the balance from proteins of this family that actively block apoptosis to those that promote apoptosis or inhibit anti-apoptotic activity may result in increased cell death, and likewise, a shift in the balance the other way may result in increased cell survival.
Accordingly, when it is desired to increase cell death by increasing apoptosis in cells under the circumstances noted above, it would be desirable to block the activity of BCL2, BCL-XL and other members of this family which suppress or inhibit apoptosis, or to increase the activity of BAX, BAK, BAD, BCL-XS and other members of this family which promote apoptosis or inhibit anti-apoptotic activities of BCL2 or BCL-XL. Likewise, when it is desired to increase cell survival in cells by decreasing apoptosis, it would be desirable to increase the activity of BCL2, BCL-XL and other members of this family which suppress or inhibit apoptosis, or to decrease the activity of apoptosis promoters of this family as noted above.
It is an object of the present invention to provide novel proteins, including isoforms, analogs, fragments or derivatives thereof which are capable of modulating the intracellular signaling pathways leading to inflammation, cell death or cell survival, this modulation being possibly via the prodomain (CARD) of the various caspases or via kinase domains of the various kinases involved in NF-κB activation. Such novel proteins of the invention would therefore possibly be direct modulators of caspase activity (cell death pathway) and/or NF-κB activation via kinase activity (cell survival pathway). Likewise, the novel proteins of the invention are possibly indirect modulators of the intracellular biological activity of a variety of other proteins involved in the inflammation, cell death or survival pathways (e.g. FAS/APO1, p55 TNF-R, p75 TNF-R, IL-1-R, MORT-1, TRADD, RIP, TRAF2, NIK, and others). Likewise, this modulation may also possibly be by direct or indirect interaction with members of the BCL2 family of proteins, the novel proteins of the present invention may be able to modulate the activity of BCL2 or other proteins of this family and in this sense the novel proteins of the invention may be indirect modulators of the various caspases, which, in turn, are modulated by members of the BCL2 family of proteins.
Another object of the invention is to provide antagonists (e.g. antibodies, peptides, organic compounds, or even some isoforms) to the above novel proteins, including isoforms, analogs, fragments and derivatives thereof, which may be used to inhibit the inflammation, cell death or survival signaling processes, when desired.
A further object of the invention is to use the above novel proteins, isoforms, analogs, fragments and derivatives thereof, to isolate and characterize additional proteins or factors, which may be involved in regulation of the inflammation, cell death or survival pathways and influence their activity, and/or to isolate and identify other receptors or other cellular proteins further upstream or downstream in the signaling process(es) to which these novel proteins, analogs, fragments and derivatives bind, and hence, in whose function they are also involved.
A still further object of the invention is to provide inhibitors which can be introduced into cells to bind or interact with the novel proteins and possible isoforms thereof, which inhibitors may act to inhibit inflammation, cell death or survival processes when desired.
Moreover, it is an object of the present invention to use the above-mentioned novel proteins, isoforms and analogs, fragments and derivatives thereof as antigens for the preparation of polyclonal and/or monoclonal antibodies thereto. The antibodies, in turn, may be used, for example, for the purification of the new proteins from different sources, such as cell extracts or transformed cell lines.
Furthermore, these antibodies may be used for diagnostic purposes, e.g. for identifying possible disorders related to abnormal functioning of cellular effects mediated directly by caspases, kinases, proteins belonging to the BCL2 family, or TRAF proteins or mediated by the p55 TNF receptor, FAS/APO1 receptor, or other related receptors and their associated cellular proteins (e.g. RAIDD, MORT-1, TRADD, RIP), which act directly or indirectly to modulate/mediate intracellular processes via interaction with TRAF proteins, caspases, kinases, or members of the BCL2 family of proteins.
A further object of the invention is to provide pharmaceutical compositions comprising the above novel proteins, isoforms, or analogs, fragments or derivatives thereof, as well as pharmaceutical compositions comprising the above noted antibodies or other antagonists.